Indole compounds and more particularly indole and skatole can be produced by sources of different type. The most important source for indole is the development of micro-organisms. Indeed, when they grow, bacteria having the tryptophanase enzyme produce indole from the tryptophan contained in a nutrient medium. For example, in the field of biochemistry and microbiology, the detection of indole is a well-known means for determining a taxonomic classification of micro-organisms. This differentiation is important firstly from an <<epidemiological>> standpoint, and secondly on account of the differences in sensitivity to antibiotics of the different bacterial strains.
In the agri-food sector, the detection of indole allows the determination of the freshness of sea produce (shrimps, shellfish, etc.). Indole can effectively be considered to be an indirect factor indicating the bacterial load of products and therefore the state of advance of the decomposition thereof. In this same field, the detection of indole is also important for the marketing of meat, in particular pork or lamb. Indeed, meat may, in some cases, have a strong sickening smell on cooking and prevent the consumption thereof. This smell derives from indole and skatole which have accumulated in the adipose tissue and the smell of which is perceived when the cooking temperature rises. The combined detection of indole and skatole in milk or meat can also be used as a mode for tracing the type of cattle feed (grass, food concentrate). In addition, studies have also shown that a high indole content in cattle feed could be a triggering factor of pulmonary emphysema.
In the field of veterinary screening, the evidencing of a large amount indole in cow milk is sufficient evidence that the cow under consideration suffers from mastitis [1].
Indole is also an active compound used in some pesticides as an attraction odour identical to the odour produced by the pheromones of some insects such as the chrysomelid corn rootworm and beetles, etc. Therefore the detection of indole must be able to be used as a way of monitoring the dose of sprayed insecticide and this, as part of health and environmental inspections. Again in the field of environmental control, and more particularly the quality of waste water derived from intensive farming, cosmetic industries or chemical industries, it is important to determine the content of indole since it is extremely toxic for aquatic organisms.
In the field of health inspections, the detection of indole and skatole may be of interest for evidencing the presence of some types of fungi (H. paupertinus, Tricholoma bufonium, T. inamoenum, T. lascivum, T. sulphureum, Boletus calopus, etc.) [2]. This could allow the classifications for example of edible or poisonous species, detections of mould in indoor air responsible for lung diseases and allergies, or even maturation tests for cheese production, indole and skatole being found in cheeses such as mozzarella, Emmenthal cheese, Hervé or Limburger cheese [3].
Finally, in the perfume, cosmetics and flavouring fields, indole is used in very low concentration as a fragrant component (flower, jasmine scent).
From the foregoing it is clear that the detection and optional quantification of indole compounds and more particularly of indole and skatole could be applied to numerous fields.
In the state of the art, colorimetric methods are already known for the analysis of indole derivatives mainly in liquid phase, and also instrumental methods are known which are rather more dedicated to gas phase identification and quantification of indole derivatives but which necessitate tedious steps of purification and preparation of samples.
Regarding the colorimetric methods, use is often made of p-dimethylaminobenzaldehyde (DMABA or DAB) and p-dimethylaminocinnamaldehyde (DMACA) as sensitive reagents for the detection of indole, skatole, tryptophan and other derivatives [4-6]. Recently, new colorimetric compounds containing an aldehyde function have been proposed for the detection of indole: p-methoxybenzaldehyde (MOB) and 4-methoxy-1-naphtaldehyde (MON) [7]. Although less used, croconic acid is also a reagent for the detection of indole. The molecular structure of the different compounds is illustrated in Scheme 1 below:

One of the major problems is the ability to assay indole, skatole or tryptophan selectively and to discriminate each of these constituents from potential interfering compounds which are often present in the same solution. The derivatives of indole effectively only differ through the type of side chain (see Scheme 2).

Volkl and Quadbeck used croconic acid to evidence the presence of indole and its derivatives in an aqueous solution containing about 60% concentrated sulfuric acid [8]. According to this article, the product of the reaction with indole absorbs at 495 nm with a coefficient of absorption ε=1.7·105 L·mol−1·cm−1; the product of skatole absorbs at 520 nm with a coefficient of absorption ε=1.8·104 L·mol−1·cm−1 and the product of tryptophan at 467 nm (coefficient of absorption not determined). It is therefore possible to assay indole and skatole quantitatively with respective detection limits of the order of 1·10−6 and 1·10−5 mol·L−1. It is also to be noted that these high coefficients of absorption restrict the dynamic measurement range over two orders of magnitude i.e. 1·10−4 and 1·10−3 mol·L−1 for indole and skatole respectively. The other probable interfering compounds noted by the authors are 2-methylindole (maximum absorption at 520 nm), and N-methylindole (maximum absorption at 495 nm) whose reaction products have coefficients of molecular absorption of the same order as those of indole and skatole.
The Ehrlich and Kovac reagents are prepared from DMABA diluted in an alcohol solution to which is added 10 to 20 volume % of concentrated hydrochloric acid. These reagents with indole afford a product of pink-red colour [4,5] whose maximum absorption is at 563 nm [6]. With 3-indolemethylpropionic, 3-indolepropionic and 3-indolebutyric acids they give a product of blue-red colour (575 nm) in solution after extraction with xylene [4] and of red-violet colour after two minutes' heating of the solution to be assayed containing a piece of resin on which a few drops of reagent have been deposited [9]. DMABA also produces a reaction with skatole and tryptophan. The reaction compound with skatole is formed rapidly, it is of blue-purple colour (λ=578 nm). Conversely, the compound formed by reaction with tryptophan is yellow in colour (λ=460 nm), the colouring develops very slowly (up to 24 hours) and the colouring is little intense compared with those given by the products of indole and skatole.
DMACA is also used for the detection of indole and its derivatives under conditions similar to those described for DMABA. DMACA affords a blue-green product (λmax=640 nm) [5] with indole and red-violet (λ=562 nm) with large quantities of tryptophan (>2.5 mg·mL−1), with indoleacetic acid and with skatole [10].
All studies are in agreement that the tests conducted with DMACA are more sensitive than those based on a reaction with DMABA. The main advantage of these techniques based on reactions in liquid phase is the ease of conducting these tests since they entail contacting the reagents, waiting for the development of colouring and obtaining a UV-visible absorption spectrum or colorimetric measurement in order to determine concentration. However, these techniques suffer from several weak points. Regents based on DMACA, croconic acid or DMABA are not stable over time, and it is therefore necessary to prepare a new reagent solution at least every month which must be stored in a cool place and away from light. Since the development of colouring is subject to change over time, it is necessary to prepare a new standard range before each of the analyses and to observe a fixed time between the test and reading. The use of colorimetric methods is not possible if the solution to be tested is already coloured (the case often encountered in biological analysis), this often requiring an extraction step of indole derivatives using a solvent (xylene, chloroform). To conclude, despite the different preparations and probe molecules used, no study has managed to find a formulation with which it is possible to obtain a test having 100% selectivity for an indole derivative in particular, i.e. which is able to achieve substantial discrimination between different compounds.
Other detection techniques consist of using reagents adsorbed on a substrate with analytes in solution. The Vracko method consists of contacting a liquid medium containing derivatives of indole with a strip of absorbent material containing DMABA and hydrochloric acid [11]. The test is positive if pink-red colouring forms on the contact surface between the paper and the liquid to be tested. As a variant of this test, the hydrochloric acid can be replaced by oxalic acid.
These tests are not intended for conducting quantitative analyses, they just allow the detection of the presence or absence of indole. These types of strips are available commercially (Sigma-Aldrich) but the use-by date is short and very strict conditions for storage are given to users. To verify that the kits are still active, the suppliers recommend performing prior tests either with indole-producing and non-indole-producing bacteria or directly with a dilute indole solution.
Finally, other detection techniques consist of using reagents adsorbed on a substrate with analytes in gas phase. For example, the study by Kohno et al. concerns the colorimetric detection of indole in gas phase [12]. They deposited a film of Nafion containing DMABA on a polyester sheet. After exposure to indole vapours, the absorption spectrum of the film exhibits a double absorption band (535 and 580 nm) and the film is of purple colour. If the film is subsequently dipped in a 6% hydrogen peroxide solution, an intense pink-violet colour develops and the absorption bands of the product move towards the UV range (480 and 535 nm). Finally, if the film is rinsed in water and dried in dry air, the spectrum again changes to show only an intense band centred on 510 nm, when the colour of the film is magenta. This colour is stable for at least 2 h. The authors left interfering compounds to appear some with 2-methylindole and 1-methylindole and with pyrrole which give a product having a spectrum similar to that of the product with indole. On the other hand, skatole, pyridine, thiophene and the ketones do not at all interfere. The aliphatic amines, aniline and furan, give a product of yellow colour which is easily distinguishable from that of indole. The detection limit of this technique is of the order of 2 ppm indole in air at relative humidity higher than 30%. The entire duration of the 3 steps of the process is approximately 50 min. The response of the sensor to an increase in concentration is not linear but rather more parabolic between 0 and 15 ppmV. The sensitivity of the sensor is relatively constant at relative humidity values higher than 30% but drops drastically at relative humidity values lower than 20%.
International application WO 2006/107370 to Mac Donald mentions the gas phase detection of indole with the reagent DMACA adsorbed on a solid substrate [13]. The various substrates described are adsorbent paper, fabrics, plastic films, microporous films (with no other specification) of silica (SiO2), of alumina, of zirconium oxide, of magnesium oxide, of titanium oxide, of iron oxide, of zinc oxide and nanoparticles of silica in powder form. The purpose in this case it is to detect the presence of indole for identifying a bacterium, but there is no quantitative measurement of the indole content in the gas phase. In addition, no study concerning interfering compounds is mentioned.
None of the conventional techniques for detecting indole is able to meet the criteria for practical analysis that is low cost, rapid, selective and sensitive. Each of the previously cited techniques has advantages and disadvantages but none meet all the criteria at the same time. In addition, none of the techniques allows gas phase or liquid phase analysis using the same device.
There is therefore a true need for a sensor at least selective for indole, that is portable (<1 kg), can be used without any particular training, capable of providing a sensitive result, of having a minimum detection limit without prior pre-concentration on the order of 1·10−6 mol·L−1, of giving a quick result (<15 min) and of covering the broadest possible range of detection (at least 3 orders of magnitude) without requiring the preparation of samples. In addition, this sensor must be able to be used indifferently for gas phase or liquid phase analyses, even by contact with solid or semi-solid samples. Finally, the response of this sensor must be stable over time and must not exhibit variability in response as a function of its storage time.